Abstract

Tuberculosis (TB) is a bacterial infectious disease. It causes 1.5 million deaths each year, 95% of which occur in low- and middle-income countries (LMICs). The disease particularly affects young adults, with adverse consequences for economic growth. In many LMICs, this problem is compounded by the problem of TB in cattle (bovine TB). This 'double-hit' is a particular problem in Ethiopia, which has the tenth highest burden of human TB in the world: bovine TB is widespread (prevalence 5-30%) and the country's economy is highly dependent on cattle farming. Improved TB control in humans and cattle would significantly enhance economic growth and welfare in Ethiopia and other similarly-burdened LMICs.

Most TB cases in humans arise when a dormant TB infection re-activates to cause disease. Dormant TB infection is very common in LMICs and this pool of infection will need to be treated if TB is ever going to be eliminated. Antibiotic treatment for dormant TB infection is effective, but roll-out of this treatment is limited by the lack of an accurate 'gold standard' diagnostic test for the condition. Existing tests for dormant TB infection rely on detecting an immune response to the bacteria, rather than detecting the bacteria itself. However, less than 10% of those with a positive immune test ever go on to develop active TB. This makes it difficult to target antibiotic treatment for dormant TB infection at the people who will benefit from it.

This situation could be about to change: very recently, a team of researchers in Germany (one of whom is a co-applicant on this grant) has reported that they have been able to detect TB bacteria in the blood of people with dormant TB infection. The number of bacteria is very small, and quite a large amount of blood (100 ml) is needed in order to detect them reliably. This remarkable finding has been made in just seven people living in Europe. The next step is to run the new test in a larger group of people who live in a country where TB is widespread. To take this idea forward, we have forged a new collaboration between Ethiopian and British scientists who together have the exact combination of clinical and technical skills needed to evaluate this new test.

We will carry out the following studies. First, we will recruit 200 healthy people who have recently been in contact with an infectious case of TB ('TB contacts') - 100 urban residents of Addis Ababa who have been exposed to a person with infectious lung TB living in their home, and 100 rural cattle herders who have been exposed to a cow suffering from bovine TB. We will run the new blood test in these TB contacts, and, where we find dormant TB bacteria in the blood stream, we will do genetic fingerprinting to see if the dormant bacteria found in the bloodstream are identical to those isolated from the case of infectious TB (human or bovine) to which they were exposed. If the genetic fingerprints of the TB bacteria are the same, this will suggest that detection of TB bacteria in the blood of people with dormant TB infection is very unlikely to be a 'chance finding'.

We will also recruit 40 people who are about to start antibiotic treatment for dormant TB infection, and run the new test before and after treatment. If we find that the antibiotic treatment clears dormant TB bacteria from the blood stream, this would suggest an additional potential application for the new test: monitoring response to antibiotic treatment of dormant TB infection. This is important, because existing tests cannot tell if antibiotic treatment for dormant TB infection has been effective or not.

Taken together, these studies will give us a clear idea as to whether the findings of the German team can be reproduced in a LMIC setting. If so, then we will apply for further funding to optimise the test to use smaller blood volumes, and conduct definitive studies to evaluate whether it could be integrated into existing TB control programmes in LMICs.

Technical Summary

Background

Efforts to identify and treat people with latent tuberculosis (TB) infection are impeded by the lack of a gold standard diagnostic test for this condition. Very recently, it has been reported that DNA of M. tuberculosis complex (MTBC) can be detected in CD34+ pluripotent haematopoietic stem cells isolated from peripheral blood of sensitised healthy TB contacts (n=7, Europe), using qRT-PCR.

We will conduct cross-sectional studies enrolling 200 asymptomatic contacts of infectious TB cases in humans and cattle. The novel qRT-PCR assay will be used to detect MTBC DNA in participants' PBMC. Where this is found, amplicon libraries will be generated and sequences compared to those of MTBC isolates from associated human / bovine index cases. We will also conduct a prospective study, in which results of the novel qRT-PCR assay will be compared pre- vs. post-chemoprophylaxis in 40 people with latent TB infection.

Laboratory techniques

MTBC-specific DNA in CD34+ PBMC will be quantitated by qRT-PCR. PCR-based methods will also be used to selectively amplify SNP-rich regions (amplicons) in MTBC genomic DNA detected in CD34+ PBMC of TB contacts. Amplicon sequences will be compared with those identified in MTBC isolated from index cases.

Application of resultsThis research could lead to the development of a more specific test for latent TB infection, with potential to revolutionise global TB control.

Planned Impact

1. Who will benefit from this research?

The following groups could potentially benefit from this research:

a) People with latent TB infection - recently or remotely acquired, living in high-, medium- or low-income settingsb) Policy-makers in the TB control community at international, national and local levelsc) The TB research community, in particular epidemiologists, clinical trialists, microbiologists and immunologistsd) Partner academic institutionse) Study participantsf) Study staff

2. How will they benefit from this research?

If results of this study show promise, and subsequent large-scale evaluations confirm a niche for this new test in TB control programmes, the primary beneficiaries will be the wider public living in high TB transmission settings: they will benefit from the development of a more specific test for latent TB infection, that could be used to target chemoprophylaxis at specific populations who stand to benefit from it.

Policy-makers in the TB control community will also stand to benefit - the study could eventually yield a new diagnostic tool for latent TB infection that could make roll-out of chemoprophylaxis much more cost-effective. Interested parties include those working internationally (e.g. WHO Stop TB Partnership and non-governmental organisations), and nationally (e.g. Ethiopian Ministry of Health). Positive results of the study will have international relevance, as TB is a global problem. For example, results of the study could have relevance to the UK, where the vast majority of cases of active TB arise as a result of reactivation of latent TB infection: if latent infection could be diagnosed with greater specificity, and response to chemoprophylaxis could be assessed, then cost-effectiveness of efforts to identify and treat latent TB infection would be improved.

Researchers in TB epidemiology, clinical trials, microbiology and immunology could also benefit from the study. Improved knowledge about transmission pathways arising from the sequencing work proposed could inform scientific efforts to develop interventions to control TB.

Several parties directly involved in the conduct of the study will also benefit. Individual participants will benefit from screening for active TB, which they would not otherwise receive. Research staff will benefit from training in research methodology, phlebotomy and computer skills, all of which are applicable in the wider healthcare or academic sectors. Participating institutions will also benefit from new UK-Ethiopian links that could be used to conduct further collaborative projects, with potential benefit to human health in both countries.

If the new qRT-PCR test shows promise in the current study, the benefits above would be likely to be realised within the medium- to long-term. The investigation we propose is designed to rapidly inform a go/no-go decision on the utility of the novel microbiological test; definitive evaluation would require larger studies with longer follow-up, which are beyond the scope of a GCRF Foundation Award. We would seek support for such studies from MRC Response Mode Schemes if the current project yielded positive results.